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Promega pcr clean up column
Pcr Clean Up Column, supplied by Promega, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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RNA Extraction:

Article Title: The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
Article Snippet: Paragraph title: RNA extraction, PCR amplification, and sequencing. ... The NA, hemagglutinin (HA), and matrix (M) genes were amplified by PCR, purified with a PCR clean-up column (Promega), sequenced (Eurofins MWG Operon), and analyzed using Lasergene 8.0 software.

Amplification:

Article Title: The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
Article Snippet: .. The NA, hemagglutinin (HA), and matrix (M) genes were amplified by PCR, purified with a PCR clean-up column (Promega), sequenced (Eurofins MWG Operon), and analyzed using Lasergene 8.0 software. .. SU101 E. coli cells with an lacZ gene regulated by an LexA operator ( ) were transformed with each pBLM TMD expression plasmid and grown overnight in lysogeny broth (LB) with 100 μg/ml ampicillin.

Polymerase Chain Reaction:

Article Title: The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
Article Snippet: .. The NA, hemagglutinin (HA), and matrix (M) genes were amplified by PCR, purified with a PCR clean-up column (Promega), sequenced (Eurofins MWG Operon), and analyzed using Lasergene 8.0 software. .. SU101 E. coli cells with an lacZ gene regulated by an LexA operator ( ) were transformed with each pBLM TMD expression plasmid and grown overnight in lysogeny broth (LB) with 100 μg/ml ampicillin.

Purification:

Article Title: The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
Article Snippet: .. The NA, hemagglutinin (HA), and matrix (M) genes were amplified by PCR, purified with a PCR clean-up column (Promega), sequenced (Eurofins MWG Operon), and analyzed using Lasergene 8.0 software. .. SU101 E. coli cells with an lacZ gene regulated by an LexA operator ( ) were transformed with each pBLM TMD expression plasmid and grown overnight in lysogeny broth (LB) with 100 μg/ml ampicillin.

Sedimentation:

Article Title: The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
Article Snippet: Virus-containing medium was harvested ∼72 h posttransfection or postinfection and clarified by sedimentation (5,000 × g , 5 min), and the supernatant was layered on top of a 20% sucrose cushion (100 mM NaCl, 10 mM Tris-Cl [pH 7.4]) and sedimented (130,000 × g , 40 min, 4°C) in a Beckman TLA 100.2 rotor. .. The NA, hemagglutinin (HA), and matrix (M) genes were amplified by PCR, purified with a PCR clean-up column (Promega), sequenced (Eurofins MWG Operon), and analyzed using Lasergene 8.0 software.

Generated:

Article Title: The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
Article Snippet: Viral RNAs were extracted from the viral pellet with the RNeasy minikit (Qiagen). cDNAs were generated with the AffinityScript multiple temperature cDNA synthesis kit (Agilent) with a primer complementary to the conserved 3′ end of the viral RNAs. .. The NA, hemagglutinin (HA), and matrix (M) genes were amplified by PCR, purified with a PCR clean-up column (Promega), sequenced (Eurofins MWG Operon), and analyzed using Lasergene 8.0 software.

Sequencing:

Article Title: The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
Article Snippet: Paragraph title: RNA extraction, PCR amplification, and sequencing. ... The NA, hemagglutinin (HA), and matrix (M) genes were amplified by PCR, purified with a PCR clean-up column (Promega), sequenced (Eurofins MWG Operon), and analyzed using Lasergene 8.0 software.

Software:

Article Title: The Influenza Virus Neuraminidase Protein Transmembrane and Head Domains Have Coevolved
Article Snippet: .. The NA, hemagglutinin (HA), and matrix (M) genes were amplified by PCR, purified with a PCR clean-up column (Promega), sequenced (Eurofins MWG Operon), and analyzed using Lasergene 8.0 software. .. SU101 E. coli cells with an lacZ gene regulated by an LexA operator ( ) were transformed with each pBLM TMD expression plasmid and grown overnight in lysogeny broth (LB) with 100 μg/ml ampicillin.

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  • 80
    Promega pcr clean up columns
    Molecular analysis of Xanthomonas strains. Total genomic <t>DNA</t> of different Xanthomonas ) or integron gene cassette <t>PCR.</t> ( Top ) BOX-PCR fingerprinting. ( Middle ) ERIC-PCR fingerprinting. ( Bottom ) Amplification of proximal integron gene cassettes by using primers MRG17 and AJH60. Samples, identified by accession number and assigned pathovar, are as follows: A, DAR30538 campestris ; B, DAR54703 begoniae ; C, DAR69819 begoniae ; D, DAR26930 vesicatoria ; E, DAR26929 translucens ; F, DAR61714 oryzae ; G, DAR73878 vitians ; H, DAR41335 vitians ; I, DAR41369 pelargoni ; J, DAR58715 pelargoni ; K, DAR61713 oryzae ; L, DAR73877 vesicatoria ; M, DAR34985 vesicatoria ; N, DAR34876 axonopodis ; O, DAR72009 pruni ; P, DAR33337 pruni ; Q, DAR56679 pruni ; R, DAR69855 unknown; S, DAR65973 citri ; T, DAR73872 citri ; U, DAR65869 citri ; V, DAR72029 citri ; W, DAR73889 citri ; and X, negative (water) control. Molecular weight markers (m) are 100-bp ladders.
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    Promega wizard pcr preps columns
    Analysis of PEVE distribution and expression in planarian body (A) Six fragments of the planarian body presented in the scheme were used to prepare first-strand cDNA and genomic <t>DNA</t> samples, which were subsequently employed for <t>PCR</t> with oligonucleotide primers for PEVE ORFs. Lines 1–6 correspond to the tissue samples taken as shown at scheme. PCR was performed with oligonucleotide primers for ORF2S (5'-caacgatagtcaccggaatgtca-3' and 5'-gcggctcctgtcttgagtcc-3') and ORF4L: (5'-ttttcatcagcatgtccgttcg-3' and 5'-cctcggttcaggcatctgtttc-3') Each PCR cycle included 95°C for 10 s, 64°C for 15 s and 72°C for 40 s. twenty one cycles were performed in the case of genomic DNA samples and twenty four cycles in the case of cDNA samples. Line 7 – negative control. RNA sample from zone 4 was used for RT-PCR without prior first-strand cDNA synthesis. (B) Northern blot analysis of the ORF2S (line 2) and ORF1L (line 3) transcripts. Line 1 – negative control. Hybridization with ORF2S probe was performed on RNA sample pre-treated with RNase A. M – marker, 0,16–1.77 kb RNA ladder (Gibco BRL).
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    Promega pcr clean up mini columns
    Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the <t>DNA</t> binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and <t>qRT-PCR</t> was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.
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    Molecular analysis of Xanthomonas strains. Total genomic DNA of different Xanthomonas ) or integron gene cassette PCR. ( Top ) BOX-PCR fingerprinting. ( Middle ) ERIC-PCR fingerprinting. ( Bottom ) Amplification of proximal integron gene cassettes by using primers MRG17 and AJH60. Samples, identified by accession number and assigned pathovar, are as follows: A, DAR30538 campestris ; B, DAR54703 begoniae ; C, DAR69819 begoniae ; D, DAR26930 vesicatoria ; E, DAR26929 translucens ; F, DAR61714 oryzae ; G, DAR73878 vitians ; H, DAR41335 vitians ; I, DAR41369 pelargoni ; J, DAR58715 pelargoni ; K, DAR61713 oryzae ; L, DAR73877 vesicatoria ; M, DAR34985 vesicatoria ; N, DAR34876 axonopodis ; O, DAR72009 pruni ; P, DAR33337 pruni ; Q, DAR56679 pruni ; R, DAR69855 unknown; S, DAR65973 citri ; T, DAR73872 citri ; U, DAR65869 citri ; V, DAR72029 citri ; W, DAR73889 citri ; and X, negative (water) control. Molecular weight markers (m) are 100-bp ladders.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Integrons in Xanthomonas: A source of species genome diversity

    doi: 10.1073/pnas.0406620102

    Figure Lengend Snippet: Molecular analysis of Xanthomonas strains. Total genomic DNA of different Xanthomonas ) or integron gene cassette PCR. ( Top ) BOX-PCR fingerprinting. ( Middle ) ERIC-PCR fingerprinting. ( Bottom ) Amplification of proximal integron gene cassettes by using primers MRG17 and AJH60. Samples, identified by accession number and assigned pathovar, are as follows: A, DAR30538 campestris ; B, DAR54703 begoniae ; C, DAR69819 begoniae ; D, DAR26930 vesicatoria ; E, DAR26929 translucens ; F, DAR61714 oryzae ; G, DAR73878 vitians ; H, DAR41335 vitians ; I, DAR41369 pelargoni ; J, DAR58715 pelargoni ; K, DAR61713 oryzae ; L, DAR73877 vesicatoria ; M, DAR34985 vesicatoria ; N, DAR34876 axonopodis ; O, DAR72009 pruni ; P, DAR33337 pruni ; Q, DAR56679 pruni ; R, DAR69855 unknown; S, DAR65973 citri ; T, DAR73872 citri ; U, DAR65869 citri ; V, DAR72029 citri ; W, DAR73889 citri ; and X, negative (water) control. Molecular weight markers (m) are 100-bp ladders.

    Article Snippet: Amplified fragments were purified by using PCR clean-up columns (Promega), and DNA was sequenced directly by using the amplification primers or cloned into pGEM-T (Promega) according to the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Amplification, Molecular Weight

    Analysis of PEVE distribution and expression in planarian body (A) Six fragments of the planarian body presented in the scheme were used to prepare first-strand cDNA and genomic DNA samples, which were subsequently employed for PCR with oligonucleotide primers for PEVE ORFs. Lines 1–6 correspond to the tissue samples taken as shown at scheme. PCR was performed with oligonucleotide primers for ORF2S (5'-caacgatagtcaccggaatgtca-3' and 5'-gcggctcctgtcttgagtcc-3') and ORF4L: (5'-ttttcatcagcatgtccgttcg-3' and 5'-cctcggttcaggcatctgtttc-3') Each PCR cycle included 95°C for 10 s, 64°C for 15 s and 72°C for 40 s. twenty one cycles were performed in the case of genomic DNA samples and twenty four cycles in the case of cDNA samples. Line 7 – negative control. RNA sample from zone 4 was used for RT-PCR without prior first-strand cDNA synthesis. (B) Northern blot analysis of the ORF2S (line 2) and ORF1L (line 3) transcripts. Line 1 – negative control. Hybridization with ORF2S probe was performed on RNA sample pre-treated with RNase A. M – marker, 0,16–1.77 kb RNA ladder (Gibco BRL).

    Journal: BMC Genomics

    Article Title: Complete genome sequence of a novel extrachromosomal virus-like element identified in planarian Girardia tigrina

    doi: 10.1186/1471-2164-3-15

    Figure Lengend Snippet: Analysis of PEVE distribution and expression in planarian body (A) Six fragments of the planarian body presented in the scheme were used to prepare first-strand cDNA and genomic DNA samples, which were subsequently employed for PCR with oligonucleotide primers for PEVE ORFs. Lines 1–6 correspond to the tissue samples taken as shown at scheme. PCR was performed with oligonucleotide primers for ORF2S (5'-caacgatagtcaccggaatgtca-3' and 5'-gcggctcctgtcttgagtcc-3') and ORF4L: (5'-ttttcatcagcatgtccgttcg-3' and 5'-cctcggttcaggcatctgtttc-3') Each PCR cycle included 95°C for 10 s, 64°C for 15 s and 72°C for 40 s. twenty one cycles were performed in the case of genomic DNA samples and twenty four cycles in the case of cDNA samples. Line 7 – negative control. RNA sample from zone 4 was used for RT-PCR without prior first-strand cDNA synthesis. (B) Northern blot analysis of the ORF2S (line 2) and ORF1L (line 3) transcripts. Line 1 – negative control. Hybridization with ORF2S probe was performed on RNA sample pre-treated with RNase A. M – marker, 0,16–1.77 kb RNA ladder (Gibco BRL).

    Article Snippet: PCR amplified DNA fragment for probe preparation was purified using Wizard PCR Preps Columns (Promega) and labeled using Prime-a-Gene Labeling System (Promega).

    Techniques: Expressing, Polymerase Chain Reaction, Negative Control, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Hybridization, Marker

    Identification of a B2-containing cathepsin S transcript. (A) RT-PCR detection of a B2-containing cathepsin S transcript in CD4 + T lymphocytes (lane 1), cultured EL4/13.3 cells (lane 2) and tumor-derived EL4/13.3 cells (lane 3). Amplification with specific β-actin primers is shown as a control for RNA-reversed transcription. (B) Scheme of the amplified B2-containing cathepsin S transcript. The open arrowhead box shows the location and orientation of the B2 element. The black box represents exon 3 of cathepsin S. PCR primers F1 and R1 are indicated. (C) Sequence analysis of the exon 3-putative intron 3 region of the cathepsin S gene and the B2 element. Sequences are aligned with exon 3 and flank of the Mus musculus cathepsin S gene (accession no. AF051727), and with published mouse B2 element (36). Boxes indicate identities. Arrows above and below the DNA sequence show the location of the forward and reverse primers used in PCR. The A and B boxes of the split polymerase III promoter and the poly (A) addition signals are underlined. The canonical splicing donor site is noted as ss. The nucleotide sequence between exons 3 and 4 of the cathepsin S gene is not available.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Tumoral Environment Triggers Transcript Anomalies in Established Tumors: Induction of Altered Gene Expression and of Aberrant, Truncated and B2 Repeat-Containing Gene Transcripts 1

    doi:

    Figure Lengend Snippet: Identification of a B2-containing cathepsin S transcript. (A) RT-PCR detection of a B2-containing cathepsin S transcript in CD4 + T lymphocytes (lane 1), cultured EL4/13.3 cells (lane 2) and tumor-derived EL4/13.3 cells (lane 3). Amplification with specific β-actin primers is shown as a control for RNA-reversed transcription. (B) Scheme of the amplified B2-containing cathepsin S transcript. The open arrowhead box shows the location and orientation of the B2 element. The black box represents exon 3 of cathepsin S. PCR primers F1 and R1 are indicated. (C) Sequence analysis of the exon 3-putative intron 3 region of the cathepsin S gene and the B2 element. Sequences are aligned with exon 3 and flank of the Mus musculus cathepsin S gene (accession no. AF051727), and with published mouse B2 element (36). Boxes indicate identities. Arrows above and below the DNA sequence show the location of the forward and reverse primers used in PCR. The A and B boxes of the split polymerase III promoter and the poly (A) addition signals are underlined. The canonical splicing donor site is noted as ss. The nucleotide sequence between exons 3 and 4 of the cathepsin S gene is not available.

    Article Snippet: Amplified DNA fragments were purified from dNTPs using Wizard PCR Preps columns (Promega) and 32 P-labeled using a random-primed DNA labeling kit (Boehringer, Mannheim, FRG) according to the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Derivative Assay, Amplification, Polymerase Chain Reaction, Sequencing

    Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the DNA binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and qRT-PCR was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.

    Journal: Cancer cell

    Article Title: Modulation of the Vitamin D3 response by cancer-associated mutant p53

    doi: 10.1016/j.ccr.2009.11.025

    Figure Lengend Snippet: Mutp53 binds VDR and augments the transcriptional response of classical VDR target genes to vitamin D3 A. SKBR3 and MCF7 cells were either grown in charcoal-stripped serum supplemented medium (-D3) or treated with 1α25(OH) 2 . B. H1299 cells were transfected with a VDR expression plasmid together with plasmids expressing either full length H175Rp53, ΔN-H175Rp53 (aa 97-393), ΔC-H175Rp53 (aa 1-292) or the DNA binding domain of H175Rp53 (DBD; aa 97-292). Total protein input levels are shown in the left panel. Cells were treated with 1α25(OH) 2 D3 for 3h and cell extracts were subjected to immunoprecipitation with either anti VDR C-20 (Santa Cruz) or PAb419 as a non-specific control (ns-Ab). Immunoprecipitated proteins were analyzed as in (A). C. SKBR3 and SW480 cells were transfected with siRNA oligonucleotides specific for p53 (p53i) or LacZ (LacZi) as negative control. After 24h, 1α25(OH) 2 D3 (D3, 100nM) was added for 24h where indicated. Cells were harvested, RNA was extracted and qRT-PCR was performed with primers specific for IGFBP3 , CYP24A1 or GAPDH . Relative levels of IGFBP3 and CYP24A1 mRNA were normalized to GAPDH . D. SKBR3 cells were transfected with either p53 siRNA (sip53) or control siRNA (si control) and treated with 1α25(OH) 2 D3 (D3;100nM, 2hours) or left untreated. Cells were subjected to chromatin immunoprecipitation with antibodies directed against either p53, VDR, p300 or non-specific antibody (ns). The precipitated DNA was subjected to Real-time qPCR amplification using sets of primers specific to a region of the CYP24A promoter spanning the VDRE. Results are presented as fold over non-specific antibody. E. MDA-MB-231 cells grown in medium containing charcoal-stripped serum, or treated with 1α25(OH)2D3 (gray bars, D3;100nM, 2 hours) or left untreated (white bars), and then subjected to chromatin immunoprecipitation with antibodies directed against p53. The immnoprecipitated chromatin was eluted and then a second IP was performed with antibodies against VDR, p300 or non specific antibody (ns Ab) as a negative control. Real-time qPCR was performed as in (D). F. RNA was prepared from the experiment shown in panel C and subjected to qRT-PCR analysis with primers specific for CYP27B . G. Immunoprecipitated DNA from experiment shown in (D) was subjected to Real-time PCR amplification with primers specific to a region of the CYP27B promoter spanning the nVDRE. Results are presented as fold over non-specific antibody. All scale bars represent +/-SD.

    Article Snippet: DNA samples were extracted using PCR clean-up mini-columns (Promega).

    Techniques: Transfection, Expressing, Plasmid Preparation, Binding Assay, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Multiple Displacement Amplification

    Mutp53 associates with VDR/RXR response elements and augments transcription from such elements A. SKBR3 cells expressing p53R175H, transfected with either siRNA for p53 (sip53) or control siRNA (sicontrol) and treated with 1α25(OH) 2 D3 (D3;100nM), were subjected to chromatin immunoprecipitation with an antibodies against p53 or VDR or with a non specific antibody as a negative control. The precipitated DNA was subjected to Real-time PCR amplification using sets of primers specific to either ChIP-control (negative control), or regions of the HIRA and TGFβ1 gene promoters spanning putative VDR/RXR response elements (VDRE). Results are presented relative to the values obtained with an aliquot of the input chromatin corresponding to 1% of the total material. The results of HIRA and TGFβ1 gene promoters are presented as enrichment fold over non specific antibody and are also normalized to values obtained for the ChIP-control results. B. Reporter plasmids were either cotransfected together with mutp53 expression plasmids (R175H or R273H) into p53-null H1299 cells (upper panel) or cotransfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) into SKBR3 cells (bottom panel). Cells were either treated with 1α25(OH) 2 . All scale bars represent +/-SD.

    Journal: Cancer cell

    Article Title: Modulation of the Vitamin D3 response by cancer-associated mutant p53

    doi: 10.1016/j.ccr.2009.11.025

    Figure Lengend Snippet: Mutp53 associates with VDR/RXR response elements and augments transcription from such elements A. SKBR3 cells expressing p53R175H, transfected with either siRNA for p53 (sip53) or control siRNA (sicontrol) and treated with 1α25(OH) 2 D3 (D3;100nM), were subjected to chromatin immunoprecipitation with an antibodies against p53 or VDR or with a non specific antibody as a negative control. The precipitated DNA was subjected to Real-time PCR amplification using sets of primers specific to either ChIP-control (negative control), or regions of the HIRA and TGFβ1 gene promoters spanning putative VDR/RXR response elements (VDRE). Results are presented relative to the values obtained with an aliquot of the input chromatin corresponding to 1% of the total material. The results of HIRA and TGFβ1 gene promoters are presented as enrichment fold over non specific antibody and are also normalized to values obtained for the ChIP-control results. B. Reporter plasmids were either cotransfected together with mutp53 expression plasmids (R175H or R273H) into p53-null H1299 cells (upper panel) or cotransfected with synthetic siRNA specific for p53 (p53i) or LacZ (LacZi) into SKBR3 cells (bottom panel). Cells were either treated with 1α25(OH) 2 . All scale bars represent +/-SD.

    Article Snippet: DNA samples were extracted using PCR clean-up mini-columns (Promega).

    Techniques: Expressing, Transfection, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Amplification